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Innate immunity against Francisella tularensis is dependent on the ASC/caspase-1 axis.

Francisella tularensis is a highly infectious gram-negative coccobacillus that causes the zoonosis tularemia. This bacterial pathogen causes a plague-like disease in humans after exposure to as few as 10 cells. Many of the mechanisms by which the innate immune system fights Francisella are unknown. Here we show that wild-type Francisella, which reach the cytosol, but not Francisella mutants that remain localized to the vacuole, induced a host defense response in macrophages, which is dependent on caspase-1 and the death-fold containing adaptor protein ASC. Caspase-1 and ASC signaling resulted in host cell death and the release of the proinflammatory cytokines interleukin (IL)-1beta and IL-18. F. tularensis-infected caspase-1- and ASC-deficient mice showed markedly increased bacterial burdens and mortality as compared with wild-type mice, demonstrating a key role for caspase-1 and ASC in innate defense against infection by this pathogen.

Animals↗

Parenteral vaccination against influenza does not induce a local antigen-specific immune response in the nasal mucosa.

The immune response in the nasal mucosa to influenza vaccination in 23 patients scheduled for tonsillectomy was studied. A statistically significant increase in influenza virus-specific serum and oral fluid antibodies was observed 7 days after vaccination. The numbers of influenza virus-specific antibody-secreting cells (ASCs) in peripheral blood also increased significantly 1 week after vaccination. The numbers of ASCs in tonsils and nasal mucosa were compared with data from a recent study of nonvaccinated volunteers. The numbers of influenza virus-specific ASCs in tonsils were significantly higher in the vaccinated group, but, surprisingly, there was no significant difference between the groups in the numbers of ASCs in nasal mucosa. This suggests that the influenza virus-specific antibodies detected in oral fluid are not produced locally in the nasal mucosa and may originate from a systemic source, indicating that the vaccination may favor a systemic immune response.

Adult↗

Enteric infections in an endemic area induce a circulating antibody-secreting cell response with homing potentials to both mucosal and systemic tissues.

Enteric infections induce a response of circulating pathogen-specific antibody-secreting cells (ASC). The expression of homing receptors (HRs) on these cells was studied in patients with diarrhea caused by Vibrio cholerae in Bangladesh, an area in which cholera is endemic. The gut HR, alpha4beta7, was expressed by approximately 80% of the ASC, indicating mucosal homing of these cells. However, the peripheral lymph node HR, L-selectin, was also expressed by approximately 80% of the ASC specific to either cholera toxin or O antigen. In earlier findings after oral immunization in nonendemic areas, alpha4beta7 has been expressed by approximately 100% and L-selectin by approximately 50% of the ASC. In comparison, the present data speak for a more systemic targeting of the immune response associated with long-lasting immunity in an endemic area. The results thus provide insight for the continued development and evaluation of vaccines.

Adolescent↗

Altered states of consciousness: review of experimental data obtained with a multiple techniques approach.

OBJECTIVES: To investigate the psychophysiologic mechanisms of an altered state of consciousness (ASC) produced via systematic mental training by correlating the results of multiple computerized bioelectrographic measurements. DESIGN: All subjects were tested, using a set of modern computerized techniques comprising digital electroencephalography, measurement of the low-frequency bilateral activity of the brain, evoked bioelectrographic signals measured by computerized Kirlian photography (otherwise called gas discharge visualization [GDV]), self-reporting by subjects, linguistic testing, and profiling of mood states. LOCATION: Sweden and Russia from 1996 to 1999. SUBJECTS: Young volunteers (61) who underwent systematic mental training for not less than 7 weeks. Members of the control group (56) were not engaged in mental training. RESULTS: All participants involved in the systematic mental training showed significant positive changes in their psychoemotional status after 7 weeks of mental training. All of the techniques showed specific changes that might be associated with an ASC in the subjects. The Kirlian (GDV) patterns showed a form of "explosive activation," which was stable, reproducible, and correlated with an ASC. This led the authors to introduce the concept of short-term activation of the induced bioelectrographic processes and enabled the properties of this ACS to be determined for the first time. There were practically no changes in the control group. CONCLUSIONS: ASC activation took place with harmonization of the biopotential field of the brain, the psychic state, and the bioenergy fields. This is attributed to changes in both the psychosomatic and psychoenergetic autoregulation. This conclusion is of vital importance for understanding what happens in systematic mental training and understanding the fundamentals of bioenergetic and psychosomatic medicine.

Adult↗

Enhanced invasion of Tax-expressing fibroblasts into the basement membrane is repressed by phosphorylated ascorbate with simultaneous decreases in intracellular oxidative stress and NF-kappa B activation.

Invasion of rat fibroblastic cells Rat-1 through Matrigel was shown to be promoted by transfection with tax gene of human T-cell leukemia virus type 1. We found that an oxidation-resistant type of vitamin C (Asc), Asc-2-O-phosphate (Asc2P), inhibited the invasion of the tax-transfected Rat-1 cells (W4 cells). Intracellular Asc (Ascin), after enzymatic dephosphorylation of administered Asc2P, was more abundant in W4 cells than in Rat-1 cells, and the ratio of dehydroascorbic acid versus Asc was increased in W4 cells but scarcely in Rat-1 cells, according to the enhanced level of intracellular reactive oxygen species (ROSin) in W4 cells. Asc2P notably repressed the increases in both ROSin and secretion of matrix metalloproteases (MMPs), but did not affect Tax protein expression in tax-transfectants. NF-kappa B activation, as evidenced by its translocation to the nucleus in W4 cells, was also repressed by Asc2P. Thus, the tax-promoted invasion together with the enhanced production of MMPs occurred with NF-kappa B activation and the increase in ROSin, both of which were effectively reduced by Asc2P. These findings indicate the therapeutic efficacy of Ascin-enriching agents for the prevention against tumor invasion in which ROSin plays a major role.

Animals↗

X gene and precore region mutations in the hepatitis B virus genome in persons positive for antibody to hepatitis B e antigen: comparison between asymptomatic "healthy" carriers and patients with severe chronic active hepatitis.

Hepatitis B virus (HBV) carriers with antibody to hepatitis e antigen comprise asymptomatic carriers (ASCs), who have low replication levels of HBV, and patients with chronic active hepatitis (CAH), who have high levels of viral replication. To investigate whether defects in the X protein might be responsible for this difference in the level of viral replication, nucleotide sequences of X and precore gene regions in serum HBV were analyzed in 19 ASCs and 9 CAH patients. All patients had a point mutation creating a stop codon in the precore region. Seventeen ASCs (87.3%) had identical mutations consisting of 4 noncontiguous 1-bp deletions or an 8-bp deletion, both of which truncate the normal X protein, whereas no CAH patient had an X gene mutation (P < .001). Thus, deletion of the X protein might be responsible for the low levels of viral replication in ASCs.

Adult↗

Evidence for a novel endocrine disruptor: the pesticide propanil requires the ovaries and steroid synthesis to enhance humoral immunity.

Steroid hormones are known to affect the humoral immune response to a variety of antigens. However, the mechanisms regulating these effects are poorly understood. The immunotoxic chemical propanil and estrogen have similar effects on the immune system including augmentation of humoral immune responses. Propanil enhances the number of phosphorylcholine (PC)-specific IgG2b, IgG3, and IgM antibody-secreting cells (ASCs) in the spleen four- to sixfold 7 days after vaccination of female C57BL/6 mice with heat-killed Streptococcus pneumoniae. Several experiments were performed to test the hypothesis that propanil increases the response via an estrogenic pathway. Ovariectomy abrogated the effect of propanil on the PC-specific ASC response. Both in vitro and in vivo assays indicate that propanil does not bind either estrogen receptor (ER) alpha or beta. Exogenous estradiol administration in ovariectomized mice failed to restore the effect of propanil on the PC response. Treatment of female mice with a pure ER antagonist, ICI 182,780, or the progesterone antagonist RU486 did not inhibit the increase in ASCs. These data suggest that estrogen and progesterone do not regulate the effect of propanil. However, complete inhibition of steroid synthesis with the gonadotropin-releasing hormone (GnRH) antagonist antide abrogated the increased response in propanil-treated mice, indicating a necessary role for steroid synthesis. Experiments in male mice demonstrated that propanil increased the number of ASCs comparable to female mice. However, orchiectomy did not inhibit this effect, suggesting that androgens do not regulate the amplification of the humoral response. These data suggest a novel role for the ovarian hormones in the regulation of the PC-specific antibody response.

Animals↗

Immortalization and characterization of a Sertoli cell line from the adult rat.

To facilitate investigations of the regulation of adult Sertoli cell function, we have established a Sertoli cell line from sexually mature Sprague-Dawley rats. The cells were immortalized with the temperature-sensitive mutant of the SV40 virus, tsA255. The tsA255 large T antigen is heat-labile and efficiently promotes propagation of cells at 33 degrees C (permissive temperature) but is inactive at 40 degrees C (nonpermissive temperature). The established clonal Sertoli cell line (ASC-17D) proliferates indefinitely at the permissive temperature. However, within 48 h at the nonpermissive temperature, cell proliferation ceases. ASC-17D cells show positive staining with antibodies to cytokeratin and vimentin, consistent with the Sertoli cell origin of these cells. Transferrin and sulfated glycoprotein (SGP)-2 mRNAs were nearly undetectable in ASC-17D cells cultured at the permissive temperature, but expression of both mRNAs was induced at the nonpermissive temperature. In contrast, SGP-1 was expressed equally at both the permissive and nonpermissive temperatures. There was no increase in either transferrin or SGP-2 with FSH or dibutyryl cAMP (db-cAMP) treatment at the permissive temperature or with FSH treatment at the nonpermissive temperature. However, the steady-state levels of both of these mRNAs were substantially increased in the presence of db-cAMP at the nonpermissive temperature. In contrast, SGP-1 mRNA was not affected by either FSH or db-cAMP. These results suggest that the ASC-17D cell line is derived from adult Sertoli cells and may be useful for the study of adult Sertoli cell function.

Animals↗

Hepatobiliary scintigraphy in the diagnosis of AIDS-related sclerosing cholangitis.

Twenty human immunodeficiency virus (HIV)-positive patients were studied who presented with right upper quadrant abdominal pain, with or without abnormal biochemical liver function tests, in whom AIDS-related sclerosing cholangitis (ASC) was suspected. The results obtained from hepatobiliary scintigraphy using 99Tcm-IODIDA were compared with data from endoscopic retrograde cholangiopancreatography (ERCP), ultrasound and histological data from liver biopsy or post mortem. 99Tcm-IODIDA was abnormal in 14 patients. Liver biopsy, ERCP or post mortem confirmed ASC in 11 patients of whom 10 had an abnormal 99Tcm-IODIDA study. Ultrasound was performed in eight of the patients with confirmed ASC but was abnormal in only five of these. One patient with mild ASC on ERCP and Kaposi's sarcoma had a normal 99Tcm-IODIDA. In HIV-positive patients with right upper quadrant pain, imaging with 99Tcm-IODIDA provides a non-invasive screening test which may help to determine those patients who should be referred for ERCP.

Acquired Immunodeficiency Syndrome↗

Obervations on subjective symptomatology, coping behavior, and medical decisions is asthma.

The Asthma Symptom Checklist (ASC), describing the subjective symptoms reported to occur during asthmatic attacks, has been developed previously. In the present study, the ASC key cluster solution was replicated and refined within a sample of 374 asthmatic inpatients. All of the original symptom categories were reporduced, including two mood categories, Panic-Fear and Irritability, a Fatigue category, and two somatic categories. Hyperventilation-Hypocapnia and Airway Obstruction. Two refinements were notable: (1) The Airway Obstruction category was empirically divided into two conceptually clear components, Dyspnea anc Congestion, and (2) three secondary mood categories, Worry, Loneliness, and Anger, were identified, which describe a continuum of mood between the polar extremes of panic and irritability. Of the symptom categories, only Panic-Fear was related to the intensity of the discharge drug regimens recommended 2 to 6 mouths after ASC administration. Panic-Fear scores were independent of pulmonary function measurements. A combined index based on pulmonary functions and panic-fear yielded the best prediction of discharge steroid regiments. Finally, those physicians rated highest in "sensitivity" to their patients by their supervisors prescribed less steroids overall, but most frequently prescribed discharge steriod regimens in relation to their patients' Panic-Fear scores. In contrast, physicians rated lower on sensitivity prescribed higher steroid regimens overall, but based these drug recommendations more cleary on objective pulmonary functioning, and not in relation to their patients' Panic-Fear scores. The results strongly suggest that the ASC Panic-Fear scale is associated with coping behaviors that importantly affect the patient's overall clinical picture by increasing the apparent severity of the asthma, thereby leading to intensified treatment. The findings stress the need to evaluate independently the objective medical condition and subjective symptomatology with its related coping behavior, in order to direct appropriate modes of therapy to each.

Adaptation, Psychological↗

Vitamin B6, vitamin C, and carpal tunnel syndrome. A cross-sectional study of 441 adults.

As part of an ongoing study of carpal tunnel syndrome (CTS) in industry, we measured plasma concentrations of pyridoxal 5'-phosphate (PLP, a measure of vitamin B6 status) and total ascorbate (ASC, a measure of vitamin C status) in 441 adult volunteers from six industries and a university exercise study. In the entire study group and in non-vitamin users (n = 218), there were no significant differences in mean plasma PLP or ASC concentrations between controls (neither symptoms nor slowing), subjects with symptoms only, subjects with median nerve slowing only, or subjects with CTS (symptoms + slowing). In male non-vitamin users (n = 137), there were significant inverse univariate associations between plasma PLP concentration and the prevalence of pain, the frequency of tingling and nocturnal awakening, and the Phalen test result. In this same subgroup, the ASC/PLP ratio was directly associated with the prevalence of pain and nocturnal awakening, and with the frequency of pain, tingling, and nocturnal awakening. In multivariate analyses, plasma ASC concentration predicted more median nerve slowing and confirmed CTS, and vitamin or vitamin interaction variables were independent predictors of 20 CTS-related outcomes. These multivariate relationships often occurred only after adjustment for age, gender, body mass index, serum alkaline phosphatase activity, or tobacco use. We conclude that there are significant relationships between plasma vitamin levels and both components of CTS (specific symptoms and median nerve slowing). The interaction between plasma PLP and ASC appears to be particularly important with respect to symptoms.

Adult↗

Atypical squamous cells of undetermined significance: human papillomavirus testing in adolescents.

OBJECTIVE: To estimate the age-stratified prevalence of oncogenic human papillomavirus (HPV) infection and to evaluate risk factors for HPV acquisition among women with atypical squamous cells of undetermined significance (ASC-US). METHODS: This was a chart review of all women with ASC-US smears who underwent oncogenic HPV testing between July 2002 and February 2004. To be eligible, HPV DNA results had to be available. Data extracted from charts included demographic information as well as certain patient characteristics historically associated with HPV acquisition or carriage. RESULTS: Of 527 eligible women with ASC-US, 357 (68%, 95% confidence interval [CI] 64-72%) tested positive for oncogenic HPV. As compared with women who tested negative, this population was significantly younger and less likely to be married. When stratified by age, 77% of the women aged younger than 20 years were positive for high-risk subtypes, compared with 58% of women aged older than 25 years (P < .01). In the multivariate analysis, young age (relative risk [RR] 1.30, 95% CI 1.14.-1.49), current smoking (RR 1.14, 95% CI 1.03-1.25) and a history of chlamydial infection (RR 1.20, 95% CI 1.09-1.32) were associated with an increased likelihood of HPV infection, while oral contraceptive use (RR 0.86, 95% CI 0.72-1.03) had a marginal protective effect. The protective effect of oral contraceptives was most pronounced among adolescent women (RR 0.57, 95% CI 0.31-1.03). CONCLUSION: Given that the rate of oncogenic HPV infection approached 80% in our population of adolescent women with ASC-US, the usefulness of HPV testing in this age group requires further investigation. LEVEL OF EVIDENCE: II-3.

Adolescent↗

Hepatitis C virus genotyping in relation to neu-oncoprotein overexpression and the development of hepatocellular carcinoma.

The distribution of hepatitis C virus (HCV) genotypes among Egyptian patients positive for anti-HCV was determined and their influence, when combined with neu-oncoprotein overexpression, on the development of hepatocellular carcinoma (HCC) was examined. The study groups included asymptomatic carriers (ASC) and patients with chronic active hepatitis (CAH) and HCC. HCV genomes were detected in the sera of 27 ASC, 29 CAH and 33 HCC patients known to have HCV infection defined by EIA and recombinant immunoblotting techniques (Inno-LiA) as well as by reverse transcriptase (RT)-PCR. The HCV genotype was determined by a reverse hybridisation technique (Inno-LiPA I and II), whereas neu-overexpression was detected by the Oncogene Science EIA Kit. Eighty-nine patients were eligible for HCV genotyping; 75 patients (84.3%) were infected with a single genotype, including 1a in 11 patients (12.4%), 1b in 2 patients (2.2%) and 2a in 10 patients (11.2%). Genotype 4 (a or c+d) was detected in 51 patients (57.3%) and only one patient had genotype 10a (1.2%). Fourteen patients (15.7%) showed mixed infection; eight of them had 1a+4 (a or c+d) and four had 2a+4 (a or c+d); the remaining two cases had 1a+2a and 1b+2a. The results revealed an increased incidence of genotype 4 in CAH and HCC patients in comparison with ASC. There was also a significant overexpression of neu-oncoprotein in CAH and HCC patients compared with ASC, which was significantly associated with subtype 4 infection. The results suggest that infection with subtype 1a and 4 HCV may be considered a risk factor for the induction of neu-overexpression and subsequent development of HCC.

Adult↗

T1764G1766 core promoter double mutants are restricted to Hepatitis B virus strains with an A1757 and are common in genotype D.

To investigate the role of pre-core and basal core promoter (BCP) mutants in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (e-CHB) in Iran, Hepatitis B virus strains from 30 patients and 42 anti-HBe-positive asymptomatic carriers (ASCs) were characterized. G1896A pre-core stop mutants, detected in 77 % of e-CHB patients and 85 % of ASCs, showed no association with virus load or aminotransferase levels. Twenty per cent of e-CHB patients and 31 % of ASCs harboured T1762A1764 mutants. When this double mutation was associated with G1757, it was linked to a higher virus load in patients than when it was associated with A1757 (10(5.2+/-1.8) vs 10(3.2+/-0.8) copies ml(-1); P=0.004). Interestingly, the most common BCP mutations were T1764 and G1766, which were present in 33 % of e-CHB patients and 29 % of ASCs. These were associated with higher virus load and aminotransferase levels compared with patients lacking core promoter mutations, although this was not significant. The T1764G1766 double mutation was only present in strains with A1757 (P<0.001), which is more frequent in strains of genotype D than in those belonging to other genotypes. On the other hand, the T1762A1764 double mutation was found more frequently in association with G1757 than with A1757. The T1762A1764 double mutation forms a binding site for hepatocyte nuclear factor 1 (HNF1), which is constrained by A1757. However, the T1764G1766 double mutant may form a binding site for HNF3. Thus, position 1757 affects the emergence of promoter double mutants and would predict a relative genotypic restriction of both the T1762A1764 and the T1764G1766 double mutants.

Adult↗

Tissue-specific expression and developmental regulation of cytochrome b561 genes in Arabidopsis thaliana and Raphanus sativus.

Ascorbate (Asc) is an essential molecule in many aspects of development and stress responses in plants and animals. Cytochromes b561 (cyts b561) are tightly coupled to Asc homeostasis. These proteins are found in mammalian tissues, where they are involved in the regeneration of Asc, serving the synthesis of catecholamine neurotransmitters, and in intestinal iron reduction. Plant genomes encode homologous membrane-associated, Asc-reducible cyts b561. The expression of these proteins in plants, however, has so far not been studied. We have now examined the expression of two Arabidopsis thaliana cyt b561-encoding genes-Artb561-1 and Artb561-2-using relative-quantitative RT-PCR and in situ hybridization (ISH) techniques. The genes show overlapping and distinct tissue- and organ-specific expression patterns. Transcripts of both genes are found in leaf epidermal cells, and expression seems to correlate with leaf maturation and cessation of cell elongation. Both genes are also expressed in the epidermal cell layer of stems and roots in the L1 layer of the shoot apex, in the vascular system of leaves, stems and roots, and in the root pericycle. In addition, Artb561-1 is expressed in the root cap, whereas Artb561-2 mRNA is found in the epidermis of lateral roots, in the root meristem, and in unfertilized ovules. These observations provide important information for the elucidation of the physiological function of cyts b561 in plants.

Journal Article↗

Stimulation of interleukin 6-like B-cell differentiation factor production in human adherent synovial cells by recombinant interleukin 1.

Abnormal production of immunoglobulin in the joint space is frequently observed in patients with rheumatoid arthritis (RA). We have previously demonstrated that adherent synovial cells (ASC) from patients with RA are involved in B-cell differentiation by their spontaneous production of B-cell differentiation factor (BCDF). The regulation of the production of this factor, however, has not yet been described. We investigated the effects of recombinant interleukin 1 alpha and beta (rIL-1 alpha and rIL-1 beta) on the production of BCDF in ASC. Increased production of BCDF was observed with increased rIL-1 concentration. Production of BCDF was detected 3 h after exposure of ASC to rIL-1 and increased throughout a 48-h culture. This BCDF, assayed on SKW6-CL4 cells, was found to share a common active site with interleukin 6. The effect of rIL-1 was almost neutralized by anti-IL-1 antibody and the addition of polymyxin B did not diminish the effect of rIL-1, indicating that rIL-1 itself stimulates ASC in vitro. These results suggest that IL-1 may play a regulatory role in the production of BCDF in synovial tissue.

Cell Adhesion↗

A comparison of the humoral and cellular immune responses at different immunological sites after split influenza virus vaccination of mice.

The spleen, bone marrow and lymph nodes are all known to be important organs for the initiation and maintenance of an immune response after vaccination. To investigate the differences and similarities in the humoral and cellular immune responses between these tissues, we vaccinated mice once or twice with the conventional human dose (15 microg HA) of influenza A (H3N2) split virus vaccine and analysed the sera and lymphocytes collected from the different sites. We found that the response of antibody secreting cells (ASC) in the lymph nodes appeared to be more transient than in the spleen, possibly because the influenza-specific IgM ASC in particular might have migrated from the lymph nodes immediately after activation. The serum antibody response was found to initially correspond with the ASC response elicited in the spleen and the lymph nodes, whereas the later serum IgG reflected the ASC response in the bone marrow. Proliferation of influenza-specific CD4(+) and CD8(+) cells was predominantly observed in the spleen and was associated with higher concentrations of cytokines than in the lymph nodes. The finding of influenza-specific CD8(+) cell proliferation in the spleen indicates that a split influenza virus vaccine may stimulate a cytotoxic T-cell response. Our results also showed that the primary response elicited a mixed Th1/Th2 profile, whereas the secondary response was skewed towards a Th2 type. Each of the three tissues had a different immunological pattern, suggesting that in preclinical vaccine studies, there is a case for investigating a range of immunological sites.

Animals↗

Selected enzyme histochemistry of Sertoli cells. 2. Adult rat Sertoli cells in co-culture with peritubular fibroblasts.

Sertoli cells and peritubular fibroblasts were collected from sexually mature Wistar rats and incubated by themselves (ASC) or in co-culture (AS/PC) for ten days with or without follicle stimulating hormone (FSH). Freshly collected cells and those of the ASC and AS/PC cultures were processed for histochemical detection of three esterases and four dehydrogenases. The relative staining intensities of azo dye and formazan reaction products were recorded for the cell cultures, co-cultures and appropriate controls. Freshly collected Sertoli cells stained heavily for lactic dehydrogenase (LDH), succinic dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G-6-PDH), non-specific esterase (Est.) and 3 beta-hydroxysteroid dehydrogenase (3 beta-OLDH). For Sertoli cells alone in culture (ASC) there was a marked decline of enzyme reaction product deposition/cell for LDH and Est. and absence of cellular staining for SDH, G-G-PDH, B-Est. and 3 beta-OLDH. The addition of FSH did not change this histochemical staining pattern for the adult cells in vitro. The presence of peritubular cells in adult Sertoli cell cultures (AS/PC) resulted in the maintenance of metabolic enzyme staining (i.e., LDH, SDH, G-6-PDH and Est.) in Sertoli cells, minimal staining for B-Est., but absence of detectable enzyme reaction product for 3 beta-OLDH. Sertoli cells co-cultured with other fibroblasts or in medium pre-conditioned with peritubular cells but not containing them stained minimally for LDH and Est. and did not generate reaction product for any of the other enzymes. The addition of FSH to the AS/PT co-culture as in the ASC cultures did not affect the enzyme histochemical staining profile of Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗